Apparatus for photometric analysis including
means for scanning

ABSTRACT

A PHOTOMETRIC ANALYSIS OF A SPECIMEN IS CARRIED OUT BY MEANS OF A SCANNING AND INTEGRATING APPARATUS WHICH COMPRISES A MAIN PHOTOTUBE, A SPECIMEN HOLDER, DRIVING MEANS FOR PRODUCING RELATIVE MOVEMENT BETWEEN THE MAIN PHOTOTUBE AND THE SPECIMEN HOLDER IN ACCORDANCE WITH A PREDETERMINED SCANNING PATH, AND AN INTEGRATOR FOR INTEGRATING THE ELECTRICAL OUTPUT SIGNAL FROM THE MAIN PHOTOTUBE. A DIAPHRAGM HAS AN APERTURE WHICH REPRESENTS A PREDETERMINED MEASURING FIELD. LAMP AND LENS MEANS ARE PROVIDED FOR PRODUCING AN IMAGE OF THE APERTURE. AN AUXILIARY PHOTOTUBE IS PROVIDED FOR REGISTERING SAID IMAGE. THE DRIVING MEANS ARE ARRANGED TO ACTU-   ATE MEMBERS FOR PRODUCING A RELATIVE MOVEMENT BETWEEN SAID IMAGE AND SAID AUXILIARY PHOTOTUBE, AND SAID RELATIVE MOVEMENT IS IN ACCORDANCE WITH THE PREDETERMINED SCANNING PATH. THE AUXILIARY PHOTOTUBE ACTUATES AN ELECTRONIC GATE WHICH SWITCHES OFF THE SIGNAL FROM THE MAIN PHOTOTUBE TO THE INTEGRATOR WHEN THE AUXILIARY PHOTOTUBE DOES NOT RECEIVE ANY LIGHT FROM THE IMAGE OF THE APERTURE.

Jul 2, 1974 x T. o. CASPERSSON ETAL Re. 28,062

APPARATUS FOR PHOTOMETRIC ANALYSIS INCLUDING MEANS FOR SCANNING OriginalFiled March 8, 1971 Fig.2

Fig. 3

INVENTOR5 MAT IAS LOHA KA United States Patent Ofiice Re. 28,062Reissued July 2, 1974 28,062 APPARATUS FOR PHOTOMETRIC ANALYSISINCLUDING MEANS FOR SCANNING Torbjiirn Oskar Caspersson, Stockholm, andCosta Mattias Lomakka, Sollentuna, Sweden, by Incentive Research andDevelopment AB, Bromma, Sweden, assignee Original No. 3,711,209, datedJan, 16, 1973, Ser. No. 121,814, Mar. 8, 1971. Application for reissueJune 4, 1973, Ser. No. 366,867 Claims priority, application Sweden, Mar.9, 1970, 3,114/70 Int. Cl. G01n 21/30 US. Cl. 356-203 4 Claims Matterenclosed in heavy brackets [1 appears in the original patent but formsno part of this reissue specification; matter printed in italicsindicates the additions made by reissue.

ABSTRACT OF THE DISCLOSURE A photometric analysis of a specimen iscarried out by means of a scanning and integrating apparatus whichcomprises a main phototube, a specimen holder, driving means forproducing relative movement between the main phototube and the specimenholder in accordance with a predetermined scanning path, and anintegrator for integrating the electrical output signal from the mainphototube. A diaphragm has an aperture which represents a predeterminedmeasuring field. Lamp and lens means are provided for producing an imageof the aperture. An auxiliary phototube is provided for registering saidimage. The driving means are arranged to actuate members for producing arelative movement between said image and said auxiliary phototube, andsaid relative movement is in accordance with the predetermined scanningpath. The auxiliary phototube actuates an electronic gate which switchesoif the signal from the main phototube to the integrator when theauxiliary phototube does not receive any light from the image of theaperture.

Our US. Pat. No. 3,446,562 discloses a method and an apparatus for thephotometric analysis of a specimen, in which the specimen is scanned ina predetermined scanning course or path covering a scanning area of aconsiderably larger size than the size of the specimen. It is acharacteristic feature of this known method that the scanning movementshall be transferred to a light spot on the screen of a cathode raytube, and that said lightspot shall be registered by means of anauxiliary phototube.

It has been found that said known method and apparatus involves somedisadvantages. For example, if the scanning area is very small, thetransfer of the scanning movement to the light-spot on the screen of thecathode ray tube is comparatively complicated. The movements of thespecimen are very small, and it is very complicated to sense said smallmovements and to transfer them to the cathode ray tube by means ofposition-sensing transducers and amplifiers.

It is the main object of the invention to prvide a less complicatedmethod and apparatus for photometric analysis. It is a particular objectof the invention to substitute a simple mechanical device for thecathode ray tube of the known apparatus.

The method of the invention is characterized in providing an illuminatedaperture of a diaphragm, said aperture representing a measuring fieldlying within the scanning area, scanning said aperture, by means of anauxiliary phototube, with a scanning movement which is in accordancewith the scanning movement of the specimen, and making the output signalfrom said auxiliary phototube switch on and switch oiT the photometricanalysis, so that only the area within the measuring field is analyzed.

The apparatus of the invention comprises a main phototube, a specimenholder, driving means for producing a relative movement between the mainphototube and the specimen holder in accordance with a predeterminedscanning path, and an integrator for integrating the electrical outputsignal from the main phototube, and is characterized by a diaphragmhaving an aperture representing a predetermined measuring field, lampand lens means for producing an image of the aperture, an auxiliaryphototube for registering the image of the aperture, members operated bythe driving means to produce a relative move ment between the image ofthe aperture and the auxiliary phototube, said relative movement beingin accordance with the predetermined scanning path, and an electronicgate, operated by the auxiliary phototube to switch oil the signal fromthe main phototube to the integrator when the auxiliary phototube doesnot receive any light from the image of the aperture.

In a preferred embodiment the means for producing a movement of theimage of the aperture of the diaphragm relative to the auxiliaryphototube comprises two mirrors situated in the path of light betweenthe aperture of the diaphragm and the auxiliary phototube, said mirrorsbeing rotatably mounted and having their axes of rotation oriented atright angles to each other. Each of said axes of rotation ismechanically connected to one of the two driving members which producethe desired scanning movement of the specimen holder. In this way thediaphragm may be stationary, whereas the image of the aperture of thediaphragm can be moved relative to the auxiliary phototube by means ofthe mirrors.

The invention will be described below with reference to the accompanyingdrawing which illustrates how the invention can be used in amicrophotometer.

FIG. 1 shows a specimen to be analyzed, a diaphragm for the delimitationof the specimen, and the scanning course as followed in the photometricanalysis.

FIG. 2 shows a microphotometer having an apparatus according to theinvention.

FIG. 3 shows the electric circuit of the apparatus disclosed in FIG. 2.

FIG. 1 shows a magnified image of a specimen consisting of five cells1-5 from a human tissue, for instance. It is desired to analyze cell No.3 to find out the quantity of light-absorbing substance in said cell.This is done by passing a light beam through the cell and analyzing theabsorption of the light beam in the cell, the degree of absorptionrepresenting the quantity of lightabsorbing substance. If, for instance,the light is monochromatic and has a wave length of 2,650 Angstromunits, the absorption represents the quantity of nucleic acids in thecell. It is preferred, for the sake of convenience, to measure thetransmission in lieu of the absorption. The transmission T is:

T 10k.c.d.

wherein c.d. is the quantity of light-absorbing substance per unit ofarea. Consequently, the transmission is an exponential function of thequantity of light-absorbing substance. The light-absorbing substance isnot evenly distributed in the cell. Therefore, it is not possible tomeasure the transmission in one single measurement by means of alight-beam covering the whole cell. It is necessary to measure on asmall area in which the light-absorbing substance is likely to be evenlydistributed, and to move said measuring area so as to cover the entirearea of the cell, and to integrate the results of said measurements overthe entire cell area. FIG. 1 illustrates such a small measuring area 6.In the analysis of cells it has been found that the measuring areashould preferably have a diameter of approximately 1 micron. Thismeasuring area 6 is moved in a scanning course as indcated by the dottedline.

FIG. 1 also illustrates the contour 7 of the desired measuring field.The measuring field delimits the cell 3 from the other cell in thescanning area. It is desired that measuring shall take place only whenthe measuring area 6 is within the contour 7. How this is done will beexplained with reference to FIGS. 2 and 3.

The apparatus disclosed in FIG. 2 comprises a microscope 9 having belowits objective 11 a holder 12 for a specimen 13. A light-source 14 sendsa light-beam through the specimen 13 and the objective 11 to a mainphototube which measures the intensity of the lightbeam. The specimenholder 12 can be moved in two directions, perpendicular to each other,by means of driving members 45, 46, so as to perform the scanningmovement represented by a dotted line in FIG. 1.

The apparatus also contains a diaphragm 36, which is situated below themain phototube 10, and which defined the size of the measuring area. Ifthe desired diameter of the measuring area is 1 micron, and if themagnification is 100, the diaphragm 36 shall have an aperture having adiameter of 0.1 mm.

The apparatus also contains an optical-mechanical device consisting of alamp 38 which illuminates a diaphragm 8 through a collecting lens 39.The aperture 7 of the diaphragm has a contour corresponding to thedesired contour of the measuring area. A lens 42 produces an image ofthe aperture 7 in the image plane of the eye-piece 18. The light fromthe aperature 7 to the lens 42 is reflected in two rotatable mirrors 40,41. The mirror 40 has its axis of rotation situated in the plane definedby the drawing, whereas the mirror 41 has its axis of rotation situatedat right angles to said plane. Preferably, both mirrors are oriented tochange the direction of the light by 90. Having passed through the lens42 the light is split up in light-splitting member 43, to the effectthat part of the light travels through the opening of a diaphragm 37 toan auxiliary phototube 22, and part of the light travels through asemi-transparent mirror 17 to the eye-piece 18. The operator can nowsee, in the eye-piece 18, an image of the specimen l3 and a superimposedimage of the aperture 7. The driving members 45, 46 for the specimenholder 12 are also connected to the mirrors 40, 41 so as to rotate saidmirrors in such a direction and at such a rate of gear that the imagesproduced in the eyepiece 18 of the specimen 13 and the aperture 7accompany each other. Consequently, the aperture 7 can be considered toperform a scanning movement relative to the auxiliary phototube 22 whichis in accordance with the scanning movement of the specimen 13 relativeto the phototube 10. The lens 42 produces a real image of the aperture 7in the plane of the diaphragm 37. The aperture of the diaphragm 37 is ofa size equal to that of the diaphragm 36.

During a measuring operation the semi-transparent mirror 17 is in theposition 17a. The plane of the diaphragm 36 now contains an image of thespecimen 13, and the plane of the diaphragm 37 contains an image of theaperture 7. These two images move synchronously during the scanningoperation. The main phototube 10 measures the intensity of the lightfrom the current area of the image of the specimen. Simultaneously, theauxiliary phototube 22 receives information whether or not the measuredvalue of said area is to be registered, depending on whether or not theimage of the aperture 7 covers the aperture of the diaphragm 37.

FIG. 3 illustrates the electrical circuit. The output signal from themain phototube 10 is transmitted through an amplifier 30 to alogarithmic amplifier 31, in which it is transformed logarithmically ina way known per se, to make its intensity directly proportional to thequantity of light-absorbing substance in the measuring area 6. Afterhaving been thus transformed logarithmically, the output signal istransmitted through an electronic gate 32 to an integrator 33 in whichit is integrated over the whole field to be measured, in a way known perse. The gate 32 is opened and closed by means of the output signal fromthe auxiliary phototube 22. This output signal is amplified in anamplifier 29. The gate 32 is opened when the auxiliary phototube 22receives light from the image of the aperture 7. If such light does notreach the auxiliary phototube 22, the gate 32 will be closed. The resultwill be indicated by an instrument 34. This result will respond to thetotal quantity of light-absorbing substance in the field to be measured,defined by the aperture 7.

The specimen 13 and the aperture 7 are simultaneously visible in theeye-piece 18 when the semi-transparent mirror is in the position 17.Therefore, the position of the diaphragm 8 and the specimen 13 can beadjusted before starting the measuring process. It is easy to place thespecimen 13 in such a position, and to give the aperture 7 such a sizeand shape, that the desired portion of the image of the specimen (inthis particular case the image of the cell 3) lies within the image ofthe aperture 7. During the measuring process the images of the specimen13 and the aperture 7, respectively, will be situated upon thediaphragms 36 and 37, respective- ]y. The position of the aperture inthe diaphragm 36 upon the image of the specimen will exactly correspondto the position of the aperture in the diaphragm 37 upon the image ofthe aperture 7. In the very moment when the measuring area 6 reaches thecontour 7, that is leaves the desired measuring field, the image of theaperture 7 will no longer cover the aperture 37. Consequently, no lightreaches the auxiliary phototube 22. The output signal from the auxiliaryphototube 22 will actuate the gate 32 to the effect that the gateswitches off the connection between the main phototube 10 and theintegrator 33. When the measuring area 6 again comes within thepredetermined measuring field, light will again reach the auxiliaryphototube 22, to the effect that the gate will switch on the connectionbetween the main phototube 10 and the integrator 33.

The illustrated apparatus operates satisfactorily when the mirrors 40,41 are turned a small angle only. A simple modification makes itpossible to move the image of the aperture along a straight line,without any distortion, also when turning the mirrors in a largerturning angle. In this modified embodiment also the mirror 40 must haveits axis of rotation situated at right angles to the plane defined bythe incident light, in the same way as the mirror 41. In order to makethe image perform two movements at right angles to each other, theplanes of incidence of the two mirrors must define an angle of The termplane of incidence" relates to the plane defined by the incident and thereflected light. This means that the light beam leaving the mirror 40must be oriented at right angles to the plane defined by the drawingillustrating FIG. 2. In order to direct said light beam towards themirror 41 a stationary mirror has to be inserted between the mirrors 40and 41 to deviate the light beam by 90.

What is claimed is:

1. A scanning and integrating apparatus for the photometric analysis ofa specimen, comprising a main phototube (10), a specimen holder (12),driving means (45, 46) for producing a relative movement between themain phototube (10) and the specimen holder (12) in accordance with apredetermined scanning path, and an integrator (33) for integrating theelectrical output signal from the main phototube (10), a diaphragm (8)having an aperture (7) representing a predetermined measuring field,lamp and lens means (38, 39, 42) for producing an image of the aperture(7), an auxiliary phototube (22) for registering the image of theaperture (7), light displacing members (40, 41) operated by the drivingmeans (45, 46) to produce a relative movement between the image of theaperture (7) and the auxiliary phototube (22), said relative movementbeing in accordance with the predetermined scanning path, and anelectronic gate (32), operated by the auxiliary phototube (22), toswitch off the signal from the main phototube (10) to the integrator(33) when the auxiliary phototube (22) does not receive any light fromthe image of the aperture (7).

2. An apparatus as claimed in claim 1, characterized in that the lightdisplacing members (40, 41) consist of two mirrors situated in the pathof light between the aperture (7) and the auxiliary phototube (22), saidmirrors being rotatably mounted and having their axes of rotationoriented at right angles to each other.

3. A scanning and integrating apparatus for the photometric analysis ofa specimen, comprising a main phototube (10), a microscope (11), aholder (12) for the specimen (13), a first light-source (14) for sendinga light-beam through said specimen and said micro scope to said mainphototube (10), an integrator (33) for integrating the electrical outputsignal from said main phototube (10), a recorder (34) for receiving theoutput signal from said integrator (33), driving means (45, 46) formoving the specimen holder (12) in a predetermined scanning pathcovering a predetermined scanning area, an auxiliary phototube (22), adiaphragm (8) having an aperture (7) defining a predetermined measuringfield within the scanning area, a second lightsource (38) for sending alight-beam through said aperture (7 to said auxiliary phototube (22),mirror means (40, 41) mounted in the path of light between said aperture(7) and said auxiliary phototube (22), said mirror means being actuatedby said driving means (45, 46) to displace the light-beam along a pathcorresponding to said scanning path, and an electronic gate (32) forreceiving the output signal from said auxiliary phototube (22) and toswitch off the connection between said main phototube (10) and saidrecorder (34) when said auxiliary phototube (22) does not receive anylight from said second light-source (38).

4. A scanning and integrating apparatus for the photometric analysis ofa specimen, comprising a main phototube (10), a microscope (11), aholder (12) for the specimen (13) a first light-source (14) for sendinga light-beam through said specimen (13) and said microscope (11) to saidmain phototube (10), an integrator (33) for integrating the electricaloutput signal from said main phototube (10), a recorder 34) forreceiving the output signal from said integrator (33), driving means(45, 46) for moving the specimen holder (12) in a predetermined scanningpath covering a predetermined scanning area, said driving meanscomprising a first driving member (45) to move the specimen holder (12)in a first direction, and a second driving member (46) to move thespecimen holder (12) in a second direction, an auxiliary phototube (22),a diaphragm (8) having an aperture (7 defining a predetermined measuringfield within the predetermined scanning area, a second light-source (38)for sending a light-beam through said aperture (7) to said auxiliaryphototube (22), a first mirror (40) mounted rotatably in the path oflight between said aperture (7 and said auxiliary phototube (22), means(47) connecting said first driving member (45) with said first mirror(40) to turn said first mirror so as to displace the lightbeam from thesecond light-source (38) to the auxiliary phototube (22) in a firstdirection corresponding to the movement in the first direction of saidspecimen holder (12), means (48) connecting said second driving member(46) with said second mirror (41) to turn said second mirror so as todisplace the light-beam from the second light-source (38) to theauxiliary phototube (22) in a second direction corresponding to themovement in the second direction of said specimen holder (12), and anelectronic gate (32) for receiving the output electrical signal fromsaid auxiliary phototube (22) and to switch oil the electricalconnection between said main photo tube (10) and said recorder (34) whensaid auxiliary phototube (22) does not receive any light from saidsecond light-source (38).

References Cited The following references, cited by the Examiner, are ofrecord in the patented file of this patent or the original patent.

UNITED STATES PATENTS 2,059,221 11/1936 Fessenden 350-7 2,932,392 4/1960Burtner et a1. 2502l7 CR 3,446,562 5/1969 Caspersson et a]. 356-226RONALD L. WIBERT, Primary Examiner L. EVANS, Assistant Examiner US. Cl.X.R. 250-219 FR

